Effect of O6-methylguanine-DNA methyltransferase gene activity on repair in human cells transformed by a regulatable ada gene.
To assess the biological role of DNA methylation at the O6 position of guanine (O6MeG) a human cell line was created that contains a regulatable gene of the O6MeG-DNA methyltransferase (MT), a repair activity that removes O6MeG adducts from the DNA. MT-deficient HeLa MR cells were transformed with an SV40-based expression vector in which the bacterial MT gene ada was put under the control of a glucocorticoid-inducible MMTV promoter. In response to dexamethasone (Dex), pSV MTV ada cells actively accumulated MT protein to reach a constant level after 10-12 h of approximately 15,000 MT molecules per cell. Co-induction by Dex and 12-O-tetradecanoylphorbol-13-acetate (TPA) further accelerated this synthesis approximately 2-fold and, as a result, higher final MT levels were achieved. The inducers were added to exponentially growing cells either before or at the time of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) exposure and the kinetics of MT synthesis was studied. MNNG affected in a dose-dependent manner (i) the loss of the pre-existing MT activity; (ii) the lag before newly synthesized MT appeared; (iii) the final level of MT accumulated by the cells; and (iv) to a lesser extent the rate of MT synthesis. In cells with a down-regulated MT gene (no inducer) even small MNNG doses lead to an irreversible loss of the pre-existing MT activity, i.e. to incomplete repair, whereas an up-regulated MT gene supported the restoration of a pool of active MT molecules in the cells, i.e. an O6MeG repair that has gone to completion. Hence, effective O6MeG repair relies not only on the pre-existing MT level, but depends to an even greater extent on the state of expression of the MT gene. The activity of the MT gene also correlated with cell survival, which confirms our earlier finding that O6MeG adducts are cytotoxic for the cell.
Waldstein
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EA
Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel.
D002460:Cell Line; D004260:DNA Repair; D003907:Dexamethasone / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D005796:Genes / Q000187:drug effects; D006367:HeLa Cells / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D008770:Methylnitrosourea / Q000378:metabolism; D008780:Methyltransferases / Q000096:biosynthesis / Q000235:genetics*; D019853:O(6)-Methylguanine-DNA Methyltransferase; D010957:Plasmids; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D008769:Methylnitronitrosoguanidine; D008770:Methylnitrosourea; D003907:Dexamethasone; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D013755:Tetradecanoylphorbol Acetate